Restriction Fragment Polymorphism Research Articles

Molecular Characterization of Liver Fluke Isolated from Sheep, Goat and Cattle in Sulaymaniyah, Iraq.

We aimed to determine species of liver fluke that predominately cause fascioliasis in sheep, goats, and cattle in the Sulaymaniyah Province, Iraq using the molecular technique of DNA sequencing and restriction fragment length polymorphism (RFLP). The samples were collected from November 2021 to May 2022. The flukes were collected from infected livers of livestock at the slaughterhouse of Sulaymaniyah Governorate, Iraq. A total of 205 flukes were collected from 56 hosts, cattle (n=22), sheep (n=28), and goats (n=6). The specific primers for FCOX1 and 28S rDNA gene amplification were used. The PCR products were subjected to restriction fragment polymorphism (RFLP) assay using Hpy188III and Dra II restriction enzymes, besides DNA sequencing. The results showed the genetic polymorphisms among the flukes. Three patterns of RFLP were observed Fasciola hepatica, F. gigantica, and F. intermediate, where 28 of them displayed F. hepatica (sheep, n=14, goat, n=3 and cattle, n= 11), whereas 24 samples displayed the F. gigantica (sheep, n=12, goat, n=3 and cattle, n= 9), and only four samples belonged to F. intermediate (sheep n=3 and cattle, n=1). In addition, the result of the ribosomal DNA (28S rDNA) sequencing confirmed that the isolated flukes belonged to F. hepatica, F. gigantica and F. intermediate. All three main species are present in the study area and F. hepatica predominated among the animal species in this area also, our results concluded that PCR-RFLP is a rapid and reliable method for liver fluke species identification.

The Effect of FSHR (G2039A) Polymorphism on Müllerian Duct Development and Hormonal Profile of Women with Primary Amenorrhea.

The function of follicle-stimulating hormone (FSH) is mediated by binding to its G-protein coupled receptor (GPCR) which is expressed on granulosa cells of the ovary. The purpose of the current study was to examine the impact of FSHR G2039A polymorphism (rs6166; Ser680Asn) on clinical and radiology profiles of women with primary amenorrhea (PA) in Gujarat, India. A total of 90 women (45 controls and 45 cases) were recruited for the study after obtaining informed consent. The DNA extraction was performed on the venous blood samples collected from the participants, followed by polymerase chain reaction (PCR). The presence of polymorphism was then analyzed using restriction fragment polymorphism (RFLP) with the BSeNI enzyme. The statistical analysis was conducted using an independent t-test, chi-square test, and ANOVA. Significance was determined by a p<0.05. Results revealed that homozygous wild type genotype was observed in 46.7% (n=21) of the control group and 11.1% (n=5) of the case group. Heterozygous genotype was observed in 33.3% (n=15) of the control group and 55.6% (n=25) of the case group (p<0.001). Homozygous mutant genotype was observed in 20% (n=9) of the control group and 33.3% (n=15) of the case group (p<0.01). The hormonal profile revealed that serum levels of FSH and luteinizing hormone (LH) were significantly higher (p<0.05) in the AA and AG genotypes compared to the GG genotypes. The FSHR rs6166 G2039A was associated with PA in women, and the A allele could be considered a causative risk factor in developing the condition.

Low Frequency of Aflatoxin Induced TP53 Gene Codon 249 Mutation in Hepatocellular Carcinoma from Egyptian Patients Living in the Nile Delta Region.

Study the frequency of codon 7 (c.747 G>T, p. R249S) mutation associated with Aflatoxin B1 (AFB1) exposure in Egyptian patients with hepatocellular carcinoma (HCC). We utilized restriction fragment polymorphism and direct sequencing to assess codon 7 mutations in 104 hepatocellular carcinomas. The expression of TP53 protein in the tumors were assessed in 44 tumors by a monoclonal rabbit antibody. We identified a single 1/104 (1%) with c.747 G>T, p. R249S variant. 28/44 (63.6%) tumors showed no or occasional (less than < 5%) nuclear staining; 9/44 (20.4%) showed mild to moderate (5-49%) and 7/44 (15.9%) showed strong ≥ 50% staining. We observed much lower frequency of TP53 gene than previously published results suggesting geographical alterations in AFB1 exposure in Egypt.

First morphometric and molecular characterization of Fasciola spp. in Northwest Tunisia.

The aim of this study was to characterize the Tunisian Fasciola spp. flukes by morphometric and molecular analyses. Flukes were collected from livers of sheep slaughtered in Sejnane slaughterhouses (Bizerte gouvernorate, Northwest Tunisia) between January and March 2021.Five morphometric parameters were determined for all the liver flukes, as follows: (i) total body length (BL), (ii) distance between ventral sucker and the tail (VS-T), (iii) distance between oral sucker and ventral sucker (OS-VS), (iv) abdomen diameter (AD), (v) tail diameter (TD) and the body length to width ratio (BL/BW). Molecular identification of the fluke specimens was carried out by polymerase chain reaction, restriction fragment polymorphism (PCR-RFLP) of a 680 bp sequence of the internal transcribes spacer 1 (ITS1) gene and by amplification, sequencing, and phylogenetic analysis of a 500 bp sequence of the ITS2 gene. Morphometric measurements showed that the mean of the total body length of the adult flukes was 21.1 ± 2.7 mm with minimum and maximum lengths of 13 and 31 mm, respectively. The PCR-RFLP analysis revealed a single profile consisting of three bands of approximately 370, 100, and 60 bp. Fasciola sequences described in the present study (GenBank numbers: OQ457027 and OQ457028) showed 99.58-100% identity to Fasciola hepatica. In conclusion, the results of this study show that molecular and phylogenetic analyses confirm the presence of a single species of F. hepatica in the Sejnane region Northwest of Tunisia. However, further studies are needed to identify the occurrence of Fasciola species in other Tunisian regions.

Association of Leukotriene A4 Hydrolase polymorphisms and risk of extrapulmonary tuberculosis

Genetic risk assessment of tuberculosis development and genotypic variation influence the gene and protein expression of leukotriene A4 hydrolase (LTA4H) at various sites of tuberculosis. The relationship between the genotype and phenotype of LATH4A was investigated in relation to the genetic risk assessment of extrapulmonary tuberculosis (EPTB). This study included 137 patients with EPTB and age-sex-matched 137 normal controls of the same ethnicity. LTA4H genotyping was performed using restriction fragment polymorphisms (RFLP) and protein level was analyzed using enzyme-linked immunosorbent assay (ELISA). The LTAH4 (rs.197833A/G) AG (codominant) and GG (recessive) genotypes showed 1.86 and 1.88-fold higher risks (p=0.0004 and p&lt;0.0001 respectively) of developing EPTB. LTA4H (rs.2660898 T/G) TG (codominant) and GG (recessive) genotypes significantly increased the risk (OR:1.69, p=0.0001 and OR;1.77 p=0.008 respectively) of EPTB. Similarly, LTA4H (rs. 2540474) AG (codominant) and GG (recessive) genotypes (OR; 1.58 95% CI:1.22- 2.04, p&lt;0.0001; OR, 1.86; 95%CI 1.37-2.52, p&lt;0.0001) increased the risk of developing ETTB. The LTB4 protein levels were significantly higher in EPTB cases (mean ± SD; 3096.10±1287.6 pg/mL) than in controls (2304.50±644.15 pg/mL) (p&lt;0.0001). LTA4H recessive variants influence protein levels (functional genetics), resulting in risk of EPTB.

Plasminogen activator inhibitor-2 and impaired fibrinolysis in pregnancy and sickle cell anemia

PurposeThis is the first study that aimed to determine antigen levels in plasma and genotypes of PAI-2 in pregnant and non-pregnant homozygous sickle cell anemia (SCA) patients.MethodsThe study subjects were all Bahraini females in the reproductive age group. The study population included 31 pregnant homozygous SS (SCA) patients. Three control groups were also studied to evaluate the effect of pregnancy and SCA on PAI-2 levels and fibrinolysis: (1) 31 healthy non-pregnant volunteers; (2) 31 cases of normal pregnancy; and (3) 20 non-pregnant SCA patients. Pregnancies were screened in the second (TM2) and third (TM3) trimesters. Global coagulation, fibrinolysis rate (euglobulin clot lysis time, ECLT), PAI-2 antigen (ELISA), and PAI-2 Ser(413)/Cys polymorphism (restriction fragment length polymorphism analysis) were determined.ResultsFeto-maternal complications were documented in both pregnancy groups. PAI-2 antigen levels were undetectable in the non-pregnant groups, but was quantifiable in both pregnant groups. Impaired fibrinolysis rate and rising PAI-2 levels with progression of pregnancy were observed in both healthy and SCA subjects. These changes were more prominent in SCA, although the rise in ECLT was less steep and PAI-2 antigen levels were not significantly different compared to normal pregnancy in the third trimester. No correlation was observed between PAI-2 genotypes and plasma antigen levels. Also, no significant difference in feto-maternal complications was found in normal (n = 25) versus SCA pregnant patients (n = 30).ConclusionsThese observations suggest that with progression of pregnancy, increasing PAI-2 levels contribute to the hypercoagulable state, particularly in SCA patients.

Liver Fluke Species Identification Isolated From Humans and Animals Using PCR-RFLP and DNA Sequencing

Fasciola species are a member of flatworms belonging to the trematodes (flukes), commonly known as liver fluke, they are extremely pathogenic parasites that affect the liver of humans and animals, nowadays, most laboratories and research facilities use molecular-based techniques for identifying and describing Fasciola species. The molecular diagnostic markers such as polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and PCR-RFLP methods are accurate and more specific than the immunological and microscopical methods. The identification of the species of liver flukes will give a new clue for the treatment and control of fascioliasis. The aim, of this study, is to find the molecular characterization of Fasciola spp. isolated from humans and animals in Sulaimani city. The flukes were isolated from humans using endoscopic techniques and from slaughtered livestock at the new slaughterhouse of Sulaimani, 48 liver flukes were collected from different hosts; human (n = 3), cattle (n = 20), sheep (n = 20), and goats (n = 5) from October 2021 to April 2022. The uinversal primers ribosomal Deoxy Ribo Nuclic Acid (rDNA) were used, then the PCR products were subjected to restriction fragment polymorphism (RFLP) assay and The PCR Product was digested with restriction enzymes DraII, also the DNA sequencing was used for the PCR product of the primer Cytochrome Oxidase subuint 1 (COX1). The results of the PCR-RFLP of the 28s rDNA show the genetic polymorphisms among the flukes and two patterns of RFLP were observed F. hepatica, and F. gigantica, also the sequence analysis of the partial gene of the COX1 showed the isolated flukes belonged to F. hepatica and F. gigantica with some genetic variation, and the result of the sequences was deposited in the Gene Bank under the following Accession numbers; F. gigantica (OP718780 and OP718781) and F. hepatica (OP718782, OP718783, and OP718784). The present study concludes thatF. hepatica and F. gigantica are both responsible for human and animal Fasciolasis in Kurdistan-Iraq, Therefore, RFLP techniques and DNA sequencing are a reliable, and differential method for species and genotyping identification of liver fluke.

The impact of Beauveria species bioinocula on the soil microbial community structure in organic strawberry plantations.

The multifunctionality of microorganisms, including entomopathogenic fungi, represents a feature that could be exploited to support the development, marketing, and application of microbial-based products for plant protection. However, it is likely that this feature could affect the composition and dynamics of the resident soil microorganisms, possibly over a longer period. Therefore, the methodology utilized to evaluate such impact is critical for a reliable assessment. The present study was performed to evaluate the impact of strains of Beauveria brongniartii and Beauveria bassiana on soil bacterial and fungal communities using an approach based on the terminal restriction fragment polymorphism (T-RFLP) analysis. Soil samples in the vicinity of the root system were collected during a 3-year period, before and after the bioinocula application, in two organic strawberry plantations. Specific primers were used for the amplification of the bacterial 16S rRNA gene and the fungal ITS region of the ribosome. Data of the profile analysis from T-RFLP analysis were used to compare the operational taxonomic unit (OTU) occurrence and intensity in the inoculated soil with the uninoculated control. With regard to the impact on the bacterial community, both Beauveria species were not fully consistently affecting their composition across the seasons and fields tested. Nevertheless, some common patterns were pointed out in each field and, sometimes, also among them when considering the time elapsed from the bioinoculum application. The impact was even more inconsistent when analyzing the fungal community. It is thus concluded that the application of the bioinocula induced only a transient and limited effect on the soil microbial community, even though some changes in the structure dynamic and frequency of soil bacterial and fungal OTUs emerged.

High genetic stability of co-circulating human adenovirus type 31 lineages over 59 years.

Type 31 of human adenovirus species A (HAdV-A31) is a significant pathogen primarily associated with diarrhoea in children but also with life-threatening disseminated disease in allogeneic haematopoietic stem cell transplant (HSCT) recipients. Nosocomial outbreaks of HAdV-A31 have been frequently described. However, the evolution of HAdV-A31 has not been studied in detail. The evolution of other HAdV types is driven either by intertypic recombination, where different types exchange genome regions, or by immune escape selection of neutralisation determinants. Complete genomic HAdV-A31 sequences from sixty diagnostic specimens of the past 18 years (2003-21) were generated, including fourteen specimens of a presumed outbreak on two HSCT wards. Additionally, twenty-three complete genomes from GenBank were added to our phylogenetic analysis as well as in silico generated and previously published restriction fragment polymorphism (RFLP) data. Phylogenetic analysis of eighty-three genomes indicated that HAdV-A31 evolved slowly with six lineages co-circulating. The two major lineages were lineage 1, which included the prototype from 1962 and nine recent isolates, and lineage 2, which split into four sublineages and included most isolates from 2003 to 2021. The average nucleotide identity within lineages was high (99.8 per cent) and identity between lineages was 98.7 and 99.2 per cent. RFLP data allowed the construction of a lower-resolution phylogeny with two additional putative lineages. Surprisingly, regions of higher diversity separating lineages were found in gene regions coding for non-structural and minor capsid proteins. Intertypic recombinations were not observed, but the phylogeny of lineage 3 was compatible with an interlineage recombination event in the fibre gene. Applying the phylogenetic analysis to the presumed nosocomial outbreak excluded two suspected transmission events and separated it into two different, simultaneous outbreaks caused by different sublineages of lineage 2. However, due to the high nucleotide identity within HAdV-A31 lineages, the proof of infection chains remains debatable. This in-depth study on the molecular phylogeny of HAdV-A31 highlights the high genetic stability of co-circulating HAdV-A31 lineages over almost six decades. It also supports the epidemiological hypothesis that HAdV-A31 circulates as an etiological agent of a childhood disease infecting immunologically naive patients without strong positive selection of immune escape variants and recombinants.

Development of a Multiplex PCR Assay for Genotyping the Fish Pathogen Piscirickettsia salmonis Through Comparative Genomics.

Piscirickettsia salmonis is a bacterial pathogen that severely impact the aquaculture in several countries as Canada, Scotland, Ireland, Norway, and Chile. It provokes Piscirickettsiosis outbreaks in the marine phase of salmonid farming, resulting in economic losses. The monophyletic genogroup LF-89 and a divergent genogroup EM-90 are responsible for the most severe Piscirickettsiosis outbreaks in Chile. Therefore, the development of methods for quick genotyping of P. salmonis genogroups in field samples is vital for veterinary diagnoses and understanding the population structure of this pathogen. The present study reports the development of a multiplex PCR for genotyping LF-89 and EM-90 genogroups based on comparative genomics of 73 fully sequenced P. salmonis genomes. The results revealed 2,322 sequences shared between 35 LF-89 genomes, 2,280 sequences in the core-genome of 38 EM-90 genomes, and 331 and 534 accessory coding sequences each genogroup, respectively. A total of 1,801 clusters of coding sequences were shared among all tested genomes of P. salmonis (LF-89 and EM-90), with 253 and 291 unique sequences for LF-89 and EM-90 genogroups, respectively. The Multiplex-1 prototype was chosen for reliable genotyping because of differences in annealing temperatures and respective reaction efficiencies. This method also identified the pathogen in field samples infected with LF-89 or EM-90 strains, which is not possible with other methods currently available. Finally, the genome-based multiplex PCR protocol presented in this study is a rapid and affordable alternative to classical sequencing of PCR products and analyzing the length of restriction fragment polymorphisms.

Molecular Systematic Investigations of Three Fin Fish Cichlid Species of Oreochromis niloticus (Nile Tilapia), Genetically Improved Farmed Tilapia (GIFT) and Astronotus ocellatus (Oscar Cichlid)

Fisheries in India contribute significantly to the total GDP of agriculture and earn significant foreign exchange. Aquaculture is playing an important role in India and is bestowed with a huge biodiversity of aquatic organisms. India ranks second in total fish production in the world. Nile tilapia has become the world’s second most popular farmed fish, after carps. Oscars is a popular aquarium fish around the world. In the present study, efforts were made to analyze the extent of divergence or similarity among three cichlid finfishes (Nile Tilapia, GIFT Tilapia, and Oscar Cichlid) using molecular biology techniques such as Random Amplification of Polymorphic DNA (RAPD) and Restriction Fragment Length Polymorphism (RFLP). The Phylogenetic tree was constructed using PhyElph software to study the evolutionary relationship between the three cichlid finfish species. The Phylogenetic or evolutionary relationship was established for the three fishes, Nile tilapia, GIFT, and Oscar cichlid, with the Phylogenetic tree. It was found that Nile tilapia and GIFT share a recent common ancestor, while Oscar cichlid does not share any evolutionary relationship with Nile tilapia and GIFT.

Contribution of common CFTR variants (M470V, T854, and Q1463) to cystic fibrosis in Tunisia: haplotype analysis.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane regulator (CFTR) protein, a chloride channel located in the epithelial cell membrane. Over than 2,000CFTR mutations have been identified, which contribute to the variety of clinical phenotypes of CF. We performed a case-control study to determine p.Met470Val (M470V), p.Thr854= (T854) and p.Gln1463= (Q1463) polymorphisms frequencies in CF patients and healthy controls and to elaborate haplotype based on these SNPs. The genotyping of M470V (exon 10), T854 (exon 14a), and Q1463 (exon 24) variants were identified using polymorphism restriction fragment length polymorphism (RFLP). Statistical difference was noted in the genotype distribution of two markers, M470V and T854, between CF and control groups. However, the Q1463 polymorphism is not identified in two studied groups. Three haplotypes were found in CF patients and controls. An exclusive association between the ancestral haplotype 1-1-2 and p.Phe508del (F508del) mutation was shown. In Tunisia, this is the first work to be interested in the analysis of M470V, T854 and Q1463 polymorphisms and haplotypes associated with the most common mutation, F508del, in the Tunisian population and worldwide.

The Impact of Interferon-γ (IFN-γ) and IFN-γ-Inducible Protein 10 (IP-10) Genes’ Polymorphism on Risk of Hepatitis C Virus–Related Liver Cirrhosis

ABSTRACT Background Today there is increasing evidence concerning the association between individual genetic polymorphisms within proinflammatory cytokines and chronic hepatitis C (CHC) severity. It has been demonstrated that polymorphisms in some genes may significantly predict HCV infected patients’ susceptibility to developing liver cirrhosis or their responsiveness to the treatment. Aim We investigated the influence of single nucleotide polymorphisms (SNPs) in Interferon (IFN-γ) and Interferon Gamma-Inducible Protein 10 (IP-10) genes on cirrhosis risk in HCV-infected patients and their association with response to various direct-acting antiviral drugs (DAAs). Methods IFN-γ (+874T/A, +2109A/G) and IP-10 (−135G/A, −1447A/G) genotypes were determined in 175 CHC Egyptian HCV patients (69 liver cirrhotic and 106 non-cirrhotic patients) using either single-stranded polymorphism polymerase chain reaction (SSP-PCR) or Restriction fragment length-PCR (RFLP-PCR) methods. Results IFN-γ + 874 TA, IP-10 − 135AA, and IP-10 − 1447AA and IP-10 − 1447GG genotypes are increased in patients developing liver cirrhosis compared to non-cirrhotic ones. Although, no statistical significance in their distribution was demonstrated, indicating the lack of association between these SNPs and liver cirrhosis susceptibility in HCV-infected patients. Haplotypes analysis between different loci on all selected genes showed a significant increase in AGGA and TAGA and a significant decrease in TGGA haplotypes in cirrhotic patients. Genotype frequencies at loci −135 and −1447 of IP-10 appeared to be in complete Linkage disequilibrium (LD) (D’ = 0.999, r2 = 0.689). Conclusion Our data support the concept that IFN-γ and IP-10 gene polymorphisms are not predictors of disease progression among Egyptian patients with HCV infection.

Genetic and histopathological characterization of Toxoplasma gondii genotypes isolated from free-range chickens reared in the metropolitan region of Rio de Janeiro state, Brazil.

This study aimed to genetically characterize Toxoplasma gondii isolates obtained from free-range chickens reared in the metropolitan region of the state of Rio de Janeiro, Brazil, and to evaluate the morbidity and histological changes associated with these isolates in mice. A mouse bioassay was used to isolate T. gondii from a pool of tissue samples (brain, heart, and thigh muscles) collected from 163 chickens. The 36 isolates obtained were genetically characterized by restriction fragment polymorphism (PCR-RFLP) analysis of the SAG1, 5'-3'SAG2, aSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3 genomic regions. Seventeen atypical genotypes were identified and nine of them were reported for the first time. All identified genotypes caused clinical signs and histological changes in mice, with the majority being associated with high cumulative morbidity (65%) and severe or very severe histological changes (76%). The exclusive identification of atypical genotypes, with a predominance of new genotypes, indicates great genetic diversity of T. gondii in the region studied. In addition, the finding that all identified genotypes caused clinical signs and often severe histological changes in mice suggests potentially relevant virulence of these strains.

Analysis on Restriction Fragment Polymorphism and Determination of Genetic Diversity of Mitochondrial DNA form SEPIA Species

Analysis on Restriction Fragment Polymorphism and Determination of Genetic Diversity of Mitochondrial DNA form SEPIA Species

Effects of Short-Term Soil Tillage Management on Activity and Community Structure of Denitrifiers under Double- Cropping Rice Field.

Soil physical and chemical characteristics, soil potential denitrification rates (PDR), community composition and nirK-, nirS- and nosZ-encoding denitrifiers were studied by using MiSeq sequencing, quantitative polymerase chain reaction (qPCR), and terminal restriction fragment polymorphism (T-RFLP) technologies base on short-term (5-year) tillage field experiment. The experiment included four tillage treatments: conventional tillage with crop residue incorporation (CT), rotary tillage with crop residue incorporation (RT), no-tillage with crop residue retention (NT), and rotary tillage with crop residue removed as control (RTO). The results indicated that soil organic carbon, total nitrogen and NH4+-N contents were increased with CT, RT and NT treatments. Compared with RTO treatment, the copies number of nirK, nirS and nosZ in paddy soil with CT, RT and NT treatments were significantly increased. The principal coordinate analysis indicated that tillage management and crop residue returning management were the most and the second important factors for the change of denitrifying bacteria community, respectively. Meanwhile, this study indicated that activity and community composition of denitrifiers with CT, RT and NT treatments were increased, compared with RTO treatment. This result showed that nirK, nirS and nosZ-type denitrifiers communities in crop residue applied soil had higher species diversity compared with crop residue removed soil, and denitrifying bacteria community composition were dominated by Gammaproteobacteria, Deltaproteobacteria, and Betaproteobacteria. Therefore, it is a beneficial practice to increase soil PDR level, abundance and community composition of nitrogen-functional soil microorganism by combined application of tillage with crop residue management.

Molecular Characterization of Fasciola spp. from Some Parts of Iran

Identification of liver flukes, Fasciola hepatica, and Fasciola gigantica by morphometric parameters is not always reliable due to the overlapping measurements. This study aimed to characterize the liver flukes of animals from different parts of Iran by the genetic markers, ITS1, and COXI. We collected flukes from infected livestock in six provinces of Iran from Sep to Nov 2016. The flukes were identified by amplification of a 680 bp sequence of ITS1 locus followed by a restriction fragment polymorphism (RFLP) assay. The genetic diversity among isolates was evaluated by amplification and sequencing of a 493 bp fragment of the COXI gene. We obtained 38 specimens from Khuzestan, 22 from Tehran, 10 from Isfahan, 10 from Mazandaran, 4 from Kurdistan, and 3 from Ardabil provinces. PCR-RFLP analysis revealed two patterns, representing F. hepatica, and F. gigantica. Fifty specimens from cattle and sheep exhibited F. hepatica pattern and 37 from the cattle, sheep, buffalo, and goat that of F. gigantica. The phylogeny based on COXI revealed two distinct clades separating F. hepatica from F. gigantica. In our phylogeny, the Iranian F. gigantica isolates showed a distinct separation from the African flukes, while grouped with the East Asia specimens demonstrating a common ancestor. The F. hepatica isolates clustered with the flukes from different parts of the world, including East Asia, Europe, and South America. The present study revealed a substantial genetic difference between F. gigantica populations of Asia and Africa, while F. hepatica isolates from different parts of the world shared high similarities.

Molecular Characterization of Giardia intestinalis Detected in Humans and Water Samples in Egypt

Giardia intestinalis is a common cause of gastrointestinal illness especially in children of developing countries. Giardia assemblages A and B are the major human infective genotypes. The present study aimed to investigate the role of water supply in the epidemiology of giardiasis via genotyping G. intestinalis detected in diarrheic children and in water samples in Egyptian rural areas. Stool samples of 100 diarrheic children, 40 drinking water samples and 10 raw water samples of canals were examined microscopically for Giardia. DNA was extracted from microscopically positive faecal samples and from all of the collected water samples. Amplification of Giardia tpi gene was performed by a nested PCR using assemblage A- and assemblage B-specific primers. Giardia gdh gene was amplified by a heminested PCR. Giardia genotypes were determined by restriction fragment polymorphism (RFLP) analysis of the amplified products. Sequencing of the amplified products was performed in two faecal and two water samples RESULTS: Giardia intestinalis was detected in 24 children, in none of the drinking water samples and in all canal water samples. Giardia sub-assemblage AII was identified in all stool and raw water samples. The RFLP pattern was confirmed in sequenced samples. The presence of the same Giardia sub-assemblage in diarrheic children and in raw water samples shows by molecular evidence the potential for waterborne dissemination of Giardia in Egypt. Further studies are needed to monitor cyst levels and infectivity of the genotype detected in water for risk assessment and management.

Root Endophytes of Coffee (Coffea arabica): Variation Across Climatic Gradients and Relationships with Functional Traits

The root microbiome of Central American coffee trees was studied from four different sites experiencing different annual temperatures and precipitation levels, sampling from plots grown conventionally and under agroforestry management (with shade trees). Total community DNA was separately extracted from roots from four trees sampled from each site/management pair and analyzed using terminal restriction fragment polymorphism analysis and also next generation sequencing (Illumina) of fungal and bacterial ribosomal amplicons. Community profiles were analyzed for site and management effects and correlations to environmental parameters and tree leaf and root economic traits. Communities of both bacteria and fungi varied with site locations, but were not impacted by management system type. They also both varied strongly with environmental parameters. Fungal communities also showed significant variation that could be attributed to plant leaf and root traits. Pooled DNA samples from each site/management regime were used to generate amplicons for next generation sequencing to determine the dominant members of the coffee root microbiome at these locations. Core bacterial genera included Pantoea, Enterobacter, and Burkholderia, while fungal core communities were dominated by members of Cladosporium, Penicillium, Exidiopsis, Trechispora, and Mycena. The potential ecological function of these microbial associates is discussed.

Responses of archaeal, bacterial, and functional microbial communities to growth season and nitrogen fertilization in rice fields

Fertilization provides excess N to soil microorganisms, thus possibly affecting soil microbial abundance, activity, and community composition during rice cultivation. The abundance and diversity of archaeal, bacterial, and functional microbial communities in rice soils upon different N fertilization regimes (no fertilizer, urea, and controlled-release fertilizer) were investigated by sampling four seasonal growth stages (seedling, tillering, vegetative, maturing) under field conditions. The abundance of bacteria was significantly affected by fertilization and seasonal time, while that of the archaea was not significantly affected. Analysis of terminal restriction fragment polymorphism (T-RFLP) of 16S rRNA genes showed no effect of N fertilization on the archaeal and bacterial community composition, but changes with plant growth time. This result was confirmed by the patterns of pyrosequencing of bacterial 16S rRNA genes. The function of the methanogenic microbial community was assayed at maturing plant growth stage by determining CH4 production rates and stable isotope fractionation in the absence and presence of methyl fluoride, an inhibitor of acetoclastic methanogenesis. N fertilization had a pronounced effect on the CH4 production rate but not on the pathway of CH4 formation. Additionally, the abundance of functional microbial communities related to CH4 and N2O emissions was measured by qPCR of functional genes. Similarly to the taxonomic composition, rice growth season showed a significant effect on the abundance of the functional microbial communities represented by the mcrA, pmoA, nirK, nirS, and nosZ genes, while N addition had usually no significant effect. A similar result was also obtained by correlation analysis between CH4 and N2O emission rates and abundances of the functional microbial gene copies. In summary, rice growth time had pronounced effects on abundance, composition, and function of microbial communities in the rice soil, while the effect of N fertilization was negligible on the level of both specific functional genes and taxonomic 16S rRNA genes.